Anti-bdca-2 antibodies, methods of making and using the same

Abstract

The application provides an anti-BDCA-2 antibody and a preparation method and application thereof, and belongs to the antibody field. The application provides a preparation method of an anti-BDCA-2 antibody, positive clones are screened by carrying out ELISA detection on hybridoma cell culture supernatant and antigen protein, and the anti-BDCA-2 antibody is prepared by subcloning the positive clones. The anti-BDCA-2 antibody comprises a heavy chain and a light chain, and the variable region of the heavy chain and the variable region of the light chain are selected from the amino acid sequences shown in SEQ ID NO:1-22. The anti-BDCA-2 antibody and related drugs provided by the application can be used for inhibiting pDC to generate IFN-alpha and preventing the excessive activation of pDC. The anti-BDCA-2 antibody generated by the application can effectively inhibit the activation of pDC cells and the generation of IFN-alpha, and has great potential in resisting virus infection and preventing certain autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to an anti-BDCA-2 antibody, its preparation method, and its application. Background Technology

[0002] Blood dendritic cell antigen 2 (BDCA-2) is a type II C lectin that plays an important role in the human immune system. [1] BDCA-2 is highly specifically expressed in plasmacytoid dendritic cells (pDCs). pDCs are a special cell type in the immune system, widely distributed in lymphatic and non-lymphatic tissues. They are potent interferon-producing cells that make important contributions to innate and adaptive immunity, playing a key role, especially in the early stages of viral infections and autoimmune diseases.

[0003] BDCA-2 expression on the surface of pDCs can regulate pDC function. As an endocytic receptor, one of the main functions of BDCA-2 is endocytosis. Through endocytosis, BDCA-2 can capture and internalize antigens, further promoting antigen presentation, which is crucial for immune responses. Therefore, BDCA-2 helps the human immune system fight pathogens by participating in antigen extraction and processing. However, unlike other endocytic receptors, BDCA-2 signaling can also inhibit pDC interferon production. When BDCA-2 is activated, it blocks interferon production by inhibiting the signaling of Toll-like receptors 7 and 9 (TLR7 and TLR9), key receptors driving interferon production in viral infections. In summary, the main functions of BDCA-2 include: participating in antigen endocytosis and processing, and making fine-tuned disease responses by inhibiting pDC responses to interferon production.

[0004] During viral infection, BDCA-2 inhibits the generation of IFN-α by pDC through the TLR9 signaling pathway. [2]It inhibits the body's response to viral infections. Furthermore, BDCA-2 plays a crucial role in autoimmune diseases. In autoimmune diseases such as systemic lupus erythematosus (SLE), elevated IFN-α levels are sometimes detected, possibly due to excessive activation of pDCs. As an inhibitor of IFN-α production, BDCA-2 may help prevent excessive autoimmune reactions, thereby avoiding the onset of autoimmune diseases. In summary, BDCA-2 plays a key role in regulating pDC responses, resisting viral infections, and preventing certain autoimmune diseases. Current research continues to explore the function and clinical applications of BDCA-2.

[0005] [1]Wilson NR, Bover L, Konopleva M, Han L, et al. CD303(BDCA-2)-apotentialnovel target for therapy in hematologic malignancies[J]. LeukLymphoma.2022Jan; 63(1):19-30.

[0006] [2]Li Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides an anti-BDCA-2 antibody, its preparation method, and its applications. This antibody and related pharmaceutical products can be used to inhibit the generation of IFN-alpha by pDCs, preventing excessive activation of pDCs. The anti-BDCA-2 antibody produced by this invention can effectively inhibit the activation of pDC cells and the generation of IFN-α, showing great potential in resisting viral infections and preventing certain autoimmune diseases.

[0008] the term:

[0009] Unless otherwise specified herein, the scientific and technical terms used herein shall have the meanings understood by one of ordinary skill in the art. Generally, the nomenclature and techniques used in relation to pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein, are well-known and frequently used in the art.

[0010] In this invention, "BDCA-2" refers to a blood dendritic cell antigen. This term includes variants, homologs, orthologs, and paralogs. For example, antibodies specific to human BDCA-2 may cross-react with BDCA-2 proteins from another species, such as monkeys, under certain conditions. In other embodiments, antibodies specific to human BDCA-2 proteins may be completely specific to human BDCA-2 proteins without cross-reacting with proteins from other species or other types, or may cross-react with BDCA-2 proteins from some other species but not all others.

[0011] In this invention, "amino acid" includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid simulants that function in a manner similar to natural amino acids. Natural amino acids are amino acids encoded by the genetic code. Amino acid analogs are those having the same basic chemical structure as naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature.

[0012] In this invention, the "complementarity-determining region" is also referred to herein as CDR. The complementary determination region disclosed herein refers to the antigen-binding site within the variable region of the heavy or light chain of the antibody, i.e., the amino acid residue in the antibody that induces antigen binding. The complementary determination regions of the light and heavy chains together constitute the antigen-binding site of the antibody. Generally, the light or heavy chain of the antibody typically contains three complementary determination regions. When the complementary determination region in this invention refers to the complementary determination region of the heavy chain, it is abbreviated as HCDR, and three different complementary determination regions are designated by numbers, such as HCDR1, HCDR2, and HCDR3. When the complementary determination region in this invention refers to the complementary determination region of the light chain, it is abbreviated as LCDR, and three different complementary determination regions are designated by numbers, such as LCDR1, LCDR2, and LCDR3.

[0013] In this invention, "antigen-binding portion," "antigen-binding domain," "antigen-binding region," or "antigen-binding site" refers to a portion of an antibody that contains amino acid residues that interact with the antigen and contribute to the antibody's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion or at least one of its CDR domains.

[0014] In this invention, "variable region" refers to the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes significantly.

[0015] In this invention, "heavy chain" refers to two longer, relatively larger, identical heavy chains (H chains) in the antibody; "light chain" refers to two shorter, relatively smaller, identical light chains (L chains) in the antibody.

[0016] In this invention, "similarity" refers to the consistency of the amino acid sequence of homologous proteins and the proportion of substituted amino acids.

[0017] In this invention, "monoclonal antibody" or "monoclonal antibody" refers to an antibody molecule product composed of a single molecule. Monoclonal antibodies exhibit specific binding specificity and affinity for a specific epitope. In this invention, murine-derived antibodies refer to antibodies secreted by murine hybrid fusion cells obtained by fusing B cells derived from immunized mice with myeloma cells.

[0018] The term "EC" in this invention 50 Also known as the half-maximal effect concentration, it refers to the antibody concentration that causes 50% of the maximum effect.

[0019] The term "EC" in this invention 50 Also known as half-inhibitory concentration, IC50 50 The value can be used to measure the ability of a drug to induce apoptosis; the stronger the induction ability, the lower the value. Of course, it can also indicate the degree of tolerance of a certain cell to a drug.

[0020] On one hand, the present invention provides a method for preparing an antibody against BDCA-2, the method comprising the following steps:

[0021] (1) Hybridoma cells were obtained by fusing mouse B cells and myeloma cells;

[0022] (2) Positive clones were obtained by ELISA detection of hybridoma cell culture supernatant and antigen protein;

[0023] (3) Positive clones were subcloned and screened to obtain monoclonal antibodies;

[0024] (4) Encode the antibody nucleotide sequence and construct the expression vector;

[0025] (5) Transfect host cells and obtain anti-BDCA-2 antibodies through host cell expression.

[0026] On the other hand, the present invention provides an antibody against BDCA-2, the antibody comprising a light chain and a heavy chain, the light chain comprising a light chain complementarity-determining region, and the heavy chain comprising a heavy chain complementarity-determining region.

[0027] The light chain complementarity-determining regions of the antibody include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 25, GAT, and SEQ ID NO: 26; the heavy chain complementarity-determining regions of the antibody include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

[0028] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, LAS, and SEQ ID NO: 31; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

[0029] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 35, WAS, and SEQ ID NO: 36; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39.

[0030] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 40, RAS, and SEQ ID NO: 41; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.

[0031] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 45, SAS, and SEQ ID NO: 46; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49.

[0032] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 50, AAS, and SEQ ID NO: 51; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.

[0033] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 55, WAS, and SEQ ID NO: 56; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59.

[0034] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 60, YSS, and SEQ ID NO: 61; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64.

[0035] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 65, SAS, and SEQ ID NO: 66; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.

[0036] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 70, RAS, and SEQ ID NO: 71; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74.

[0037] Alternatively, the light chain complementarity-determining regions of the antibody may include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 75, GTS, and SEQ ID NO: 76; the heavy chain complementarity-determining regions of the antibody may include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79.

[0038] Specifically, the variable region of the light chain of the antibody includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

[0039] Specifically, the heavy chain variable region of the antibody includes SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

[0040] Specifically, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2;

[0041] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4;

[0042] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 5, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 6;

[0043] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 8;

[0044] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 10;

[0045] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 11, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 12;

[0046] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 13, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14;

[0047] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 16;

[0048] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 18;

[0049] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 19, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 20.

[0050] Alternatively, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 21, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 22.

[0051] Specifically, the light chain of the antibody is formed by splicing a light chain variable region and a light chain constant region; the heavy chain is formed by splicing a heavy chain variable region and a heavy chain constant region.

[0052] More specifically, the variable region of the light chain includes a constant region.

[0053] Preferably, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 80.

[0054] More specifically, the heavy chain variable region includes IgG1, IgG2, IgG3, IgG4 or IgG1-YTE heavy chain constant region.

[0055] Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81.

[0056] Specifically, the light chain amino acid sequence of the antibody is SEQ ID NO: 82, and the heavy chain amino acid sequence is SEQ ID NO: 83;

[0057] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 84, and the heavy chain amino acid sequence is SEQ ID NO: 85;

[0058] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 86, and the heavy chain amino acid sequence is SEQ ID NO: 87;

[0059] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 88, and the heavy chain amino acid sequence is SEQ ID NO: 89;

[0060] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 90, and the heavy chain amino acid sequence is SEQ ID NO: 91;

[0061] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 92, and the heavy chain amino acid sequence is SEQ ID NO: 93;

[0062] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 94, and the heavy chain amino acid sequence is SEQ ID NO: 95;

[0063] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 96, and the heavy chain amino acid sequence is SEQ ID NO: 97;

[0064] Alternatively, the light chain amino acid sequence of the antibody is SEQ ID NO: 98, and the heavy chain amino acid sequence is SEQ ID NO: 99;

[0065] Alternatively, the light chain amino acid sequence of the antibody may be SEQ ID NO: 100, and the heavy chain amino acid sequence may be SEQ ID NO: 101.

[0066] Alternatively, the light chain amino acid sequence of the antibody may be SEQ ID NO: 102, and the heavy chain amino acid sequence may be SEQ ID NO: 103.

[0067] On the other hand, the present invention provides a nucleic acid that encodes the aforementioned antibody.

[0068] In another aspect, the present invention provides an expression vector that encodes the aforementioned nucleic acid.

[0069] In another aspect, the present invention provides a host cell, wherein the host cell includes the above-mentioned expression vector.

[0070] In another aspect, the present invention provides the application of the above-mentioned antibody in the preparation of drugs.

[0071] Specifically, the drug exerts its effect by binding to BDCA-2 with an anti-BDCA-2 antibody, promoting BDCA-2 endocytosis, inhibiting excessive activation of pDC, and inhibiting the generation of IFN-α by pDC.

[0072] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described anti-BDCA-2 antibody, the above-described nucleic acid, the above-described expression vector, and / or the above-described host cell.

[0073] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0074] The technical effects achieved by this invention are as follows:

[0075] (1) The anti-BDCA-2 antibody and related drugs provided by the present invention can be used to inhibit the generation of IFN-α by pDC and prevent the over-activation of pDC.

[0076] (2) The anti-BDCA-2 antibody produced by this invention can effectively inhibit the activation of pDC cells and the generation of IFN-α, and has great potential in resisting viral infection and preventing certain autoimmune diseases. Attached Figure Description

[0077] Figure 1 This is the binding of anti-BDCA-2 antibody to human BDCA-2.

[0078] Figure 2This demonstrates the binding of anti-BDCA-2 antibody to monkey BDCA-2.

[0079] Figure 3 This describes the binding of anti-BDCA-2 antibody to RPMI8226-hBDCA2 cells.

[0080] Figure 4 For the detection of endocytosis of anti-BDCA-2 antibody.

[0081] Figure 5 Anti-BDCA-2 antibody inhibits TLR-ligand-stimulated PBMC (Donor1) IFN-α secretion.

[0082] Figure 6 Anti-BDCA-2 antibody inhibits TLR-ligand-stimulated PBMC (Donor2) IFN-α secretion.

[0083] Figure 7 Anti-BDCA-2 antibody inhibits TLR-ligand-stimulated PBMC (Donor2) IFN-α secretion. Detailed Implementation

[0084] The present invention will be described below with reference to specific embodiments. These embodiments are not intended to limit the present invention, but only to illustrate the present invention and make the technical solution of the present invention easier to understand and master. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0085] The instruments used in this invention are as follows:

[0086] 1. Microplate reader: Instrument manufacturer: Thermo Fisher; Instrument model: Multiskan FC.

[0087] 2. Flow cytometer: Manufacturer: Coulter Beckman; Model: CytoFLEX S.

[0088] 3. High-content imaging: Manufacturer: PerkinElmer; Instrument model: Operetta CLS.

[0089] Basic Experiment Example 1: Mouse Immunotherapy and Hybridoma Fusion

[0090] Prepare an emulsion (200 μL / mouse) containing equal volumes of PBS and Freund's complete adjuvant containing 50 μg of antigen: Connect syringes containing antigen and adjuvant respectively using a three-way valve. Push the syringe back and forth, allowing the contents to flow from one side of the syringe to the other, for 10 minutes until a stable emulsion is obtained. Inject the emulsion subcutaneously at multiple sites on the back of 6-8 week old Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and procured and bred by Suzhou Gemma Gene Co., Ltd.), immunizing a total of 5 mice. Two weeks later, prepare an emulsion (200 μL) containing equal volumes of PBS and incomplete Freund's adjuvant containing 25 μg of antigen and inject it subcutaneously at multiple sites again. Two weeks later, give the mice a booster immunization, and collect blood samples 7 days after booster immunization to measure antibody titers. When the antibody titer is sufficiently high (≥1 / 50000), administer an emulsion containing...

[0091] 25 μg of antigen in PBS solution was administered subcutaneously via the tail vein for booster immunization. Three days after booster immunization, the spleens of two mice were dissected, and splenic lymphocytes were isolated and fused with SP2 / 0 cells for hybridoma fusion.

[0092] Example 1: Molecular screening using BDCA-2 protein

[0093] Recombinant human BDCA-2 (purchased from Suzhou Nearshore Protein Technology Co., Ltd., NP: PLKAA-1, LOT: 20220816) and recombinant monkey BDCA-2 (purchased from Suzhou Nearshore Protein Technology Co., Ltd., NP: PLRBB, LOT: 20211220) were used as antigens to coat high-affinity ELISA plates overnight at 4°C, with a coating volume of 50 ng per well. The plates were blocked with 2% BSA at 37°C for 2 h. 100 μL of hybridoma supernatant was incubated with recombinant human BDCA-2 and recombinant monkey BDCA-2 for 1 h, respectively, followed by washing three times with washing buffer. Freshly diluted enzyme-labeled antibody was added, and the plates were incubated at 37°C for 30 min, followed by washing. 100 μL of TMB substrate solution was added, and the plates were incubated for 5–10 min, followed by stopping the reaction with 50 μL of 2M sulfuric acid. The absorbance at 450 nm was read using an ELISA reader. If the reading was 2.1 times greater than the OD value of the negative control, it was considered a clone that was positive for recombinant human BDCA-2 or recombinant monkey BDCA-2.

[0094] Fresh hybridoma cell pellets were collected from positively bound clones, with a cell count of 5 × 10⁶ cells. 6 More than one sample was flash-frozen in liquid nitrogen and then transported on dry ice to Suzhou Genewiz Biotechnology Co., Ltd. for monoclonal antibody sequencing. Molecules with complete variable region sequences were obtained, as shown in Table 1 below.

[0095] Table 1 Different cloning sequences

[0096]

[0097]

[0098] Example 2: Construction and expression of anti-BDCA-2 antibody

[0099] The light chain variable region and light chain constant region (SEQ ID NO: 80) of the sequenced monoclonal antibody were directly spliced ​​together to form the light chain, and the heavy chain variable region and heavy chain constant region (SEQ ID NO: 81) of the sequenced monoclonal antibody were directly spliced ​​together to form the heavy chain. The light chain and heavy chain of the antibody have the amino acid sequences shown in SEQ ID NO: 82 and SEQ ID NO: 83, respectively.

[0100] Or the amino acid sequences shown in SEQ ID NO: 84 and SEQ ID NO: 85;

[0101] Or the amino acid sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87;

[0102] Or the amino acid sequences shown in SEQ ID NO: 88 and SEQ ID NO: 89;

[0103] Or the amino acid sequences shown in SEQ ID NO: 90 and SEQ ID NO: 91;

[0104] Or the amino acid sequences shown in SEQ ID NO: 92 and SEQ ID NO: 93;

[0105] Or the amino acid sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95;

[0106] Or the amino acid sequences shown in SEQ ID NO: 96 and SEQ ID NO: 97;

[0107] Or the amino acid sequences shown in SEQ ID NO: 98 and SEQ ID NO: 99;

[0108] Or the amino acid sequences shown in SEQ ID NO: 100 and SEQ ID NO: 101;

[0109] Or the amino acid sequences shown in SEQ ID NO: 102 and SEQ ID NO: 103;

[0110] Based on the amino acid sequences of the light and heavy chains, codon optimization was performed according to human host cells. Genes were synthesized conventionally (the amino acid sequences of the light chain constant region and heavy chain constant region are shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively). The genes were cloned into the pTT5 vector (ampicillin-resistant) using 5'EcoRI and 3'HindIII. Clones were selected for sequencing, and cells with correct sequencing results were preserved and cultured for expansion. The expanded cells were used for plasmid extraction. Following the same gene synthesis and vector construction methods described above, plasmids expressing control antibodies were obtained, where the heavy chain of the control antibody is shown in SEQ ID NO: 109, and the light chain is shown in SEQ ID NO: 110.

[0111] Table 2. Control antibody sequences

[0112]

[0113]

[0114] The extracted plasmids were transfected into cells and the proteins were isolated and purified as follows:

[0115] 1. HEK-293F cells were cultured in Free Style™ 293 expression medium (Gibco, Cat#: 12338-018). Cell density was measured, and viability was greater than 95%. Cells were collected by centrifugation, and the cell density was adjusted to 5 × 10⁶ cells / year using a cell counter. 6 Cells / mL.

[0116] 2. Take a 50mL centrifuge tube, add 10% MEM of the transfection system, add each plasmid, mix well, filter, and let stand for 5 min. Add PEI (DNA to PEI ratio of 1:3, the amount of DNA added per mL of cell culture medium is 1.5 μg) to the DNA suspension, mix gently (gently invert 2-3 times), and let stand for 15-20 min. Then gently add the complex to the aliquoted cells, gently shaking the flask while adding. Place the transfected HEK-293F cells in a 37℃, 5% CO2 incubator and culture at 120 rpm. After 10-12 days of culture, collect the supernatant.

[0117] 3. Anti-BDCA-2 antibody was enriched and purified using a pre-equilibrated protein-G affinity column (GE, Cat#: 17040501) and eluted with elution buffer (20 mM citric acid, pH 3.0–3.5). The anti-BDCA-2 antibody was then incubated in PBS at pH 7.0, and antibody concentration was detected using NanoDrop.

[0118] Example 3: Cross-binding of anti-BDCA-2 antibody with human and monkey BDCA-2.

[0119] To determine whether the anti-BDCA-2 antibody cross-binds with human and monkey BDCA-2, ELISA was performed using recombinant human and monkey BDCA-2 proteins. The specific steps are as follows:

[0120] Recombinant human BDCA-2 and recombinant monkey BDCA-2 were used as antigens to coat high-affinity ELISA plates overnight at 4°C, with a coating amount of 50 ng per well. The plates were then blocked with 2% BSA at 37°C for 2 h. 100 μL of serially diluted antibodies of various concentrations were incubated with each of the recombinant human BDCA-2 and recombinant monkey BDCA-2 for 1 h, followed by washing three times with washing buffer. Freshly diluted ELISA-labeled antibody was added, and the plates were incubated at 37°C for 30 min, followed by washing. 100 μL of TMB substrate solution was added, and after 5-10 minutes of color development, 50 μL of 2M sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was read using an ELISA reader, and a binding curve was plotted, as shown in the figure. Figure 1 , Figure 2 As shown. Calculate EC 50 EC50 values ​​for each antibody 50 The values ​​are shown in Table 2.

[0121] Table 2. Anti-BDCA-2 antibody binding to human and monkey BDCA-2 protein.

[0122]

[0123]

[0124] Example 4: Anti-BDCA-2 antibody binds to human BDCA-2

[0125] To determine whether the anti-BDCA-2 antibody binds to human BDCA-2 expressed on the cell surface, FACS cell binding assays were performed using RPMI8226 cells stably overexpressing human BDCA-2 (purchased from Fuheng Biotechnology, catalog number FH0092).

[0126] 10 in 100 μL of culture medium 5 RPMI 8226 cells were plated in 96-well V plates, and 50 μL of serially diluted anti-BDCA-2 antibodies of various concentrations were added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBST. 1000-fold diluted PE-goat anti-human IgG (purchased from BioLegend, catalog number 366904) was added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBS, and cell fluorescence was detected using flow cytometry. Binding curves were plotted. Figure 3 As shown. Calculate EC 50 EC50 values ​​for each antibody 50 The values ​​are shown in Table 3.

[0127] Table 3. Anti-BDCA-2 antibody binding to human BDCA-2

[0128]

[0129]

[0130] Example 5: Detection of endocytosis of anti-BDCA-2 antibody

[0131] RPMI8226 cells stably overexpressing human BDCA-2 were used for endocytosis detection via FACS and high-content imaging. In short, Zenon... TM pHrodo TM iFL Green Human IgG Labeling Reagent was used to label the test antibody. RPMI8226-hBDCA2 cells and the labeled antibody were then incubated at 37°C for 16 hours. Cells were collected and washed. High-content imaging was used to capture the results, as shown below. Figure 4 As shown in the figure. Fluorescence intensity was detected by flow cytometry, and the results are shown in Table 4.

[0132] Table 4. Flow cytometry detection of anti-BDCA-2 antibody via endocytosis

[0133] Antibody MFI 12D5B12 2619 1C11F3 2405 4H6B5 1863 9H6C7 699 20B2C1 721 4A2C12 2352 14E6A12 1785 4B3F5 2645 5H6F9 3414 6G5H6 397 11G9B3 2735 PC 2272 iRed 224 Blank 73.7 Isotype 256

[0134] Antibodies 12D5B12, 1C11F3, 4H6B5, 9H6C7, 20B2C1, 4A2C12, 14E6A12, 4B3F5, 5H6F9, and 11G9B3 exhibited significant red fluorescence. Compared to the control group, these anti-BDCA-2 antibodies showed significant endocytosis activity.

[0135] Example 6: Anti-BDCA-2 antibody inhibits TLR-ligand-stimulated PBMC IFN-α secretion.

[0136] In this embodiment, human peripheral blood lymphocytes (PBMCs, purchased from Shanghai Saili Biotechnology Co., Ltd., catalog number XFB-HP050B) were used as the research object. PBMCs were stimulated to secrete IFN-α using ODN2216. Simultaneously with ODN2216 stimulation, a 5-fold serially diluted anti-BDCA-2 antibody was added to the plated cells. Cells containing ODN2216 and different concentrations of the test antibody were incubated overnight (18 hours) at 37°C in a cell culture incubator. Cell supernatant was collected, and the IFN-α content in the supernatant was detected using a commercially available Human IFN-α ELISA Kit (purchased from absin, catalog number abs51025-96T). Inhibition curves were plotted, and the results are shown below. Figure 5-7 As shown. Calculate IC 50 The IC50 values ​​of each antibody are shown in Table 5.

[0137] Table 5. Anti-BDCA-2 antibody IC50 50 value

[0138]

[0139] The above results indicate that the anti-BDCA-2 antibody of the present invention can effectively inhibit the activation of pDC cells and the generation of IFN-α.

[0140] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. An antibody against BDCA-2, characterized in that, The antibody comprises a light chain and a heavy chain, wherein the light chain includes a light chain complementarity-determining region and the heavy chain includes a heavy chain complementarity-determining region. The LCDR1, LCDR2, and LCDR3 of the light chain complementarity-determining regions of the antibody are shown in SEQ ID NO: 75, GTS, and SEQ ID NO: 76; the HCDR1, HCDR2, and HCDR3 of the heavy chain complementarity-determining regions of the antibody are shown in SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO:

79.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 21, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

22.

3. The antibody according to claim 1, characterized in that, The light chain of the antibody is formed by splicing together a light chain variable region and a light chain constant region; the heavy chain is formed by splicing together a heavy chain variable region and a heavy chain constant region.

4. The antibody according to claim 3, characterized in that, The light chain variable region includes the κ constant region; the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 or IgG1-YTE heavy chain constant region.

5. A nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1-4.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid as described in claim 5.

7. A host cell, characterized in that, The host cell includes the expression vector of claim 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-BDCA-2 antibody according to any one of claims 1-4, the nucleic acid according to claim 5, the expression vector according to claim 6, and / or the host cell according to claim 7.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition exerts its effects by binding to BDCA-2 with an anti-BDCA-2 antibody, promoting BDCA-2 endocytosis, inhibiting pDC overactivation, or inhibiting pDC IFN-α production.

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